JPH08269A - Fusion genes, recombinant plasmids, transformants and mutagenicity test methods - Google Patents

Abstract

(57) [Summary] [Purpose] A mutagenicity test method that detects the extent of damage to DNA by a mutagenic substance as the intensity of luminescence. It is a simple method that does not require disruption of microbial cells. We propose a test method that can detect the presence or absence of mutagenicity with high sensitivity by operation in a short time. [Constitution] An operator ( lexA operator) to which the lexA protein binds as a repressor and
fusion gene luxAB genes are attached encoding luciferase downstream of the promoter of the recA gene (recA promoter) to (lexA operator - - recA promoter luxAB gene) transformants recombinant plasmid has been introduced with the analyte After culturing using the medium containing the luciferase, a luminescent element that serves as a substrate for luciferase is added to emit light.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel and useful fusion gene, a recombinant plasmid having this fusion gene, a transformant into which this recombinant plasmid has been introduced, and mutagenicity using this transformant. Regarding test methods.

[0002]

2. Description of the Related Art Since most of the mutagenic substances are also carcinogenic substances, measuring the mutagenic substances in the environment is becoming one of the important items for safety evaluation.
However, because there are various chemical substances in the environment,
It is impossible to separate and quantify individual mutagens by instrumental analysis. Therefore, a mutagenicity test method using an organism is required so that the mutagenicity of cellular DNA can be comprehensively evaluated.

To date, many mutagenicity test methods have been developed and various biological materials have been used. Among them, the mutagenicity test method for measuring the presence or absence of mutagenicity of a sample using a microorganism has the advantage that it can be easily performed with a short time and inexpensive material, and several test methods have already been developed. -It is widely used. One of the most widely used mutagenicity test methods in the world today is the Ames-test.
There is. This test method determines the presence or absence of mutagenicity of a specimen based on the number of colonies of revertant mutants that appear when a histidine-requiring Salmonella mutant strain is applied and cultured on a plate-containing minimal medium containing the specimen. Is. However, this test method has a problem that a sample containing histidine cannot be used and that the culture takes a relatively long time (48 hours or more).

In addition to the Ames test, the SOS chromo test (for example, Japanese Patent Laid-Open No. 5-211865) and the um test (umu-test, Mutation R)
es. , 147: 219-229 (1985)) and the like. These mutagenicity test methods are test methods using a microorganism transformed by introducing a fusion gene consisting of "SOS gene that senses a mutagenic substance" and "a gene encoding a quantifiable enzyme". , SfiA or umuDC as the SOS gene, and lac that encodes β-galactosidase as the gene encoding the enzyme
Z is used. In addition, in these mutagenicity test methods, sfiA or umuDC was induced in the SOS-induced state.
The presence or absence of mutagenicity is detected by measuring the activity of β-galactosidase induced under the control of A.

Since these mutagenicity test methods use the frequency at which the SOS gene that directly senses DNA damage is read as an index of mutagenicity, they are more direct and essential than the Ames test that counts revertant colonies. Target. However, measurement of enzyme activity requires manipulation of cell disruption with a surfactant or toluene, and a large number of reagents such as o-nitrophenyl-β-D-galactopyranoside, which is a substrate of the enzyme. There is a problem that is necessary.

[0006] As another test method, Microtox (Microt) using luminescence of microorganisms as an index of mutagenicity is used.
ox) test method and mutatox test method. However, the former is a test method in which the decrease in the amount of luminescence of luminescent bacteria is used as an index of mutagenicity, and conversely, the latter uses a mutant strain of luminescent bacteria that has lost the luminescence ability, and Is a test method in which the increase in
In principle, this is different from the present invention in which the frequency of reading a gene that directly senses DNA damage is used as an index of mutagenicity.

By the way, in Japanese Patent Publication No. 4-503003, a gene system ( m
er -operon) and a bacterial luciferase gene ( luxAB ) that imparts bioluminescence, and a method for detecting mercury using a microorganism transformed with a plasmid vector containing a fusion gene. In this method, mer- specifically induced by mercury ion
The lux AB is translated by an operon to produce luciferase, and mercury is detected or quantified by the presence or absence or intensity of luminescence by this enzyme. However, the above publication does not suggest the use of luminescence by luciferase in a mutagenicity test.

[0008]

The object of the present invention is to detect the degree of damage of DNA caused by a mutagenic substance as the intensity of luminescence, and thereby to detect the mutagenicity of various compounds. It is to provide a novel and useful fusion gene that can be used for determining the presence or absence. Another object of the present invention is to provide a recombinant plasmid incorporating the above fusion gene. Another object of the present invention is to provide a transformant in which the above recombinant plasmid is introduced into a host bacterium. Still another object of the present invention is to use the above transformant, without the need for cell disruption, with a simple operation, and a mutagenicity capable of highly sensitively determining the presence or absence of mutagenicity in a short time. It is to propose a test method.

[0009]

The present invention provides the following fusion gene, recombinant plasmid, transformant and mutagenicity test method. (1) A fusion gene characterized in that a luxAB gene encoding luciferase is bound downstream of an operator to which a lexA protein as a repressor binds and a promoter of a recA gene. (2) A recombinant plasmid comprising a fusion gene having a luxAB gene encoding luciferase bound downstream of an operator to which the lexA protein as a repressor binds and a promoter of the recA gene. (3) A recombinant plasmid having a fusion gene in which a luxAB gene encoding luciferase is bound downstream of an operator to which a lexA protein binds as a repressor and a promoter of a recA gene, and which has the following restriction enzyme map (1 ) Is a recombinant plasmid pMAS10.

Embedded image (In the map, lexA op is the operator to which the lexA protein binds, recA pr is the promoter of the recA gene, luxAB is the luxAB gene, Amp is the ampicillin resistance gene, and pBR322 is the plasmid pB.
The base sequence derived from R322 is shown. (4) A transformant having the recombinant plasmid according to (2) or (3) introduced thereinto. (5) A method for testing mutagenicity, which comprises culturing the transformant according to (4) above in a medium containing a sample, and then adding a luminescent element as a substrate for luciferase to cause luminescence.

The operator constituting the fusion gene of the present invention is a binding site to which the lexA protein as a repressor binds (hereinafter sometimes referred to as lexA operator or lexA op). The nucleotide sequence of the lexA operator is known, and the existing position 1..20 of the sequence described in SEQ ID NO: 1 in the sequence listing corresponds to this.

The nucleotide sequence of the promoter of the recA gene (hereinafter, sometimes referred to as recA promoter or recA pr) constituting the fusion gene of the present invention is known, and the position of the sequence of SEQ ID NO: 19. Corresponds to this. As shown in SEQ ID NO: 1, recA
The recA promoter is regulated by the repressor because the promoter sequence and the lexA operator sequence partially overlap. lexA operator and re
You not have to necessarily duplicate the cA promoter, re by the lexA protein is a repressor
It suffices that the cA promoter be in a positional relationship so that it can be regulated. For example, a DNA fragment in which the lexA operator is present downstream of the recA promoter can also be used in the present invention.

The nucleotide sequence shown in SEQ ID NO: 1 is obtained from a commercially available plasmid or a commercially available chromosomal DNA of a microorganism,
It can be obtained by cutting out the portion containing the recA promoter with a restriction enzyme. In this case, SEQ ID NO:
Although other bases are bound before and after the base sequence described in 1., such a fragment can be used as it is as a DNA fragment containing a lexA operator and a recA promoter. It is possible to use the one with the base removed or the addition of another base in some cases. That is, the lexA operator used in the present invention has a position of SEQ ID NO: 1.
The base sequence shown in 1..20 is an essential minimum unit, and a DNA fragment in which other bases are bound before and after this sequence can also be used as a lexA operator. Also recA
The promoter also has an essential minimum unit of the nucleotide sequence shown at the position of existence 19.25 in SEQ ID NO: 1. A DNA fragment in which other nucleotides are bound before and after this sequence can also be used as the recA promoter.

A specific method for obtaining the nucleotide sequence shown in SEQ ID NO: 1 is, for example, a 236 bp fragment obtained by cutting pKH502 plasmid (commercially available from ATCC under the catalog number of No. 37293) with Eco RI and Bam HI. It can be obtained as a fragment. Alternatively, after purifying the chromosomal DNA of Escherichia coli K-12 strain (commercially available from ATCC under the catalog number No. 23716), Bam HI is cleaved to obtain a DNA fragment of about 3.1 kb. This fragment can be obtained as a Pst I-cut DNA fragment of about 1.2 kb.

LuxAB constituting the fusion gene of the present invention
Gene is a gene that luxB gene is bound downstream of luxA gene. The luxAB gene is a gene encoding a luciferase enzyme, and the luxA gene and the luxB gene each encode a luciferase subunit. luxA gene and lu
The base sequence of the xB gene is known and is described in the sequence of SEQ ID NO: 2. That is, the position 137..1201 of SEQ ID NO: 2 is the luxA gene, and the position 123 is
1..2202 is the luxB gene.

The DNA fragment containing the luxAB gene shown in SEQ ID NO: 2 can be cut out from the chromosomal DNA of a commercially available microorganism. In this case, before the luxA gene,
between the luxA gene and luxB genes, after luxB genes, although other bases than luxA gene and luxB genes are attached, it can be directly used as a luxAB genes such DNA fragments.
In addition, a part or all of other bases may be removed by enzymatic treatment, or a base to which another base is added may be used. This point is the lexA operator and r
The same as for the ecA promoter.

Specific methods for obtaining the DNA fragment shown in SEQ ID NO: 2 include, for example, ATCC No. 33
The chromosomal DNA of Vibrio harveyi commercially available under the catalog number of 843.
After purification to give a DNA fragment of about 3.9kb was cut with Hin dIII, it can be obtained as DNA fragment of about 2.4kb obtained by cutting the fragment with Sal I and Pvu II.

The fusion gene of the present invention has the above-mentioned le.
It is a fusion gene in which the luxAB gene as described above is bound downstream of the xA operator and the recA promoter. By introducing a recombinant plasmid having such a fusion gene into a host bacterium, the transformant of the present invention can be obtained.

In such a transformant of the present invention, the lexA protein as a repressor is usually lexA.
It is bound to the operator and the expression of the luxAB gene is suppressed. However, due to the mutagenic substance, DN
When A is damaged and becomes SOS-induced, the protein produced by recA activates the self-degrading ability (protease activity) of the repressor bound to the lexA operator, and the repressor protein autolyzes. In such an SOS-induced state, rec
Translation of the luxAB gene downstream of the A promoter is initiated and luciferase is produced. When a luminophore (luciferin) which is a substrate of luciferase is added thereto, luminescence is generated.

Since the intensity of luminescence has a positive correlation with the concentration of the mutagenic substance, by measuring the intensity of luminescence,
Alternatively, the presence or absence of a mutagenic substance or the relative high or low concentration can be detected by the presence or absence of luminescence. Thus, lexA operator and recA promoter acts as a sensor for sensing the mutagens, l
The uxAB gene acts as a luminescent source that converts the perceived mutagenicity into readily detectable light.

The recombinant plasmid of the present invention contains the above fusion gene, but has a DNA region capable of autonomous replication in order to exist as a plasmid in the host bacterium. Further, it is preferable to provide a drug resistance gene as a marker for detecting microorganisms during cloning, and for example, ampicillin resistance gene, chloramphenicol resistance gene, kanamycin resistance gene, tetracycline resistance gene, etc. can be used. The recombinant plasmid pMAS10 represented by the above restriction enzyme map (1) is a recombinant plasmid that can be preferably used to obtain a transformant to be used in the mutagenicity test.

The recombinant plasmid pMAS10 can be obtained as follows. First, from the Escherichia coli K-12 strain, the lexA operator and
obtaining a DNA fragment of about 1.2kb including recA promoter (Bam HI~ Pst I fragment). At this time, this fragment was cloned into the plasmid pTrc99A using Bam HI and P
Prefer incorporated into about 4.2kb of the plasmid was cut with st I. pTrc99A is No. It is commercially available from Pharmacia under the code number 27-5007-01. Next, a plasmid containing the above 1.2 kb DNA fragment was added to Pst.
After cutting with I, the cut surface is blunted with T4 polymerase and then the Sal I linker is ligated. This gives a plasmid of about 5.4 kb.

Apart from the above-mentioned operation, an approximately 2.4 kb DNA fragment ( Sal I to Pv containing the luxAB gene from Vibrio harvey (ATCC33843) was prepared by the above method.
uII fragment) is obtained. Next, after ligating Sal I linkers to the Pvu II cleavage end, was cut with Sal I, to obtain a DNA fragment of about 2.4kb which both ends are cut with Sal I.

This about 2.4 kb DNA fragment is mixed with the above-mentioned about 5.4 kb plasmid, which has been digested with Sal I and then dephosphorylated, and ligated. By this operation, the fusion gene of the present invention is created. The obtained plasmid was used as Bam HI and Hi.
by digestion with n dIII (breakpoint Hin dIII are in close proximity to the Sal I cleavage point), about 3.6 of the present invention
A kb fusion gene is obtained.

The fusion gene of about 3.6 kb obtained as described above was transformed with pBR322 plasmid into EcoRI and B.
By mixing with a plasmid cut with am HI,
The desired recombinant plasmid pMAS10 is obtained. In addition, pBR322 is No. It is commercially available from Toyobo Co., Ltd. under the code number of DNA-003.

The introduction of the recombinant plasmid into the host bacterium can be easily carried out by an electroporation method, a method of treatment with calcium and rubidium, a method of transfer using a helper plasmid, or the like. As a host bacterium, the recombinant plasmid is stably maintained and S
Bacteria having OS repair ability can be used. Such host bacteria include a series of enterobacteria such as Sal.
monella typhimurium , Alca
ligenes faecalis , Escheri
chia coli , Enterobacter c
loacae , Yersinia rukkeri and the like, among which Salmonella typhimurium ( S
mutant strain prepared from Almonella typhimurium ), for example, Fermentation Research Institute (INST
ITUTE FOR FERMENTATION, OS
No. from AKA (IFO). Salmonella typhimurium marketed under the catalog number 14209 is preferred.

To carry out a mutagenicity test using the transformant obtained as described above, the transformant is cultured in a medium containing a sample for which the presence or absence of mutagenicity is to be determined, and luciferase It can be carried out by adding a luminescent element as a substrate and causing it to emit light. The type of mutagenic substance to be detected is not limited, and examples thereof include methyl methanesulfonate, mitomycin C, and 4-nitroquinoline-N- in a sample.
Oxide, nalidixic acid, 9-aminoacridine, 2
-Nitrofluorene, N-ethyl-N'-nitro-nitrosoguanidine or mixtures thereof or mutagens which have not been known so far.

Culturing is carried out under conditions suitable for transformants. For example, Salmonella typhimurium (IFO142).
In the case of 09), it is preferably carried out at a constant temperature of 37 ° C. in a nutrient medium generally used for culturing Escherichia coli, such as LB medium. The medium may be liquid medium or solid medium, but liquid medium is preferred. A culture time of 12 to 16 hours is appropriate.

Examples of the luminescent element added after culturing in this manner include long-chain aldehydes such as n-decylaldehyde and n-nonylaldehyde. When the sample contains a mutagenic substance, since luciferase is produced as described above, the luminescent element is decomposed and luminescence is generated. By detecting this luminescence, it can be used as an index for determining the presence or absence of mutagenicity.

In a control test containing no mutagenic substance, no luminescence is generated or only a relatively weak luminescence is generated. Therefore, the presence or absence of luminescence or the change in luminescence intensity indicates the presence or absence of mutagenicity or the extent of DNA damage. Can be determined. For example, when the luminescence intensity is higher than that of the control, it can be determined that the DNA damage is that much. Usually, when the emission intensity is twice or more that of the control, it can be determined that there is mutagenicity. Luminescence is preferably carried out under the condition that the activity of luciferase is kept high, usually at a temperature of 30 to 3
It is preferable to carry out under conditions of 7 ° C. and pH around 7.

Luminescence can be easily measured by a device such as a luminometer. In this case, since the culture medium after culture can be directly subjected to luminescence measurement, crushing of bacteria is not required, and thus the test operation is simple. Further, luminescence can be quantified even in a small amount, and the frequency at which a gene that directly senses DNA damage is read is used as an index of mutagenicity, so that a mutagenic substance can be detected with high sensitivity.

[0031]

EFFECTS OF THE INVENTION According to the fusion gene of the present invention, when DNA is damaged by a mutagenic substance, the extent of damage can be detected by the intensity of luminescence, and therefore it can be used for a mutagenicity test. . Further, the recombinant plasmid of the present invention,
It can be used as a vector when preparing a transformant containing the above fusion gene and used for a mutagenicity test.

The transformant of the present invention retains the above recombinant plasmid and can be used for a mutagenicity test. Furthermore, according to the mutagenicity test method of the present invention, by using the above transformant, it is possible to carry out a mutagenicity test with high sensitivity in a short time without requiring cell disruption, with a simple operation. .

[0033]

EXAMPLES Next, examples of the present invention will be described. The plasmids and microorganisms used in the examples are as follows. 1) Escherichia coli K-12 strain ( Escheric
hia coli k-12 strain) ATCC No. ATCC with catalog number 23716
(AmericanType Culture Col
It is commercially available from L. LexA operator into the chromosomal DNA of the Escherichia coli, contains the recA promoter and recA genes, Bam
It is known that it is excised as a DNA fragment of about 3.1 kb by HI (Proc. Natl. Acad.
Sci. , 77: 313-317 (1980)).

[0034] 2) Vibrio Harubei (Vibrio
harveyi ) ATCC No. ATCC with catalog number 33843
The commercially available Vibrio hull bay was used. The chromosomal DNA of the cells includes a luxAB gene has been found to be cut out as a DNA fragment of about 4kb by Hin dIII (Biochemis
try23: 3663-3667 (1984)).

3) Escherichia coli
Hia coli ) HB101 Bethesda Research, Inc. is commercially available under the trade name of Library Efficiency ^™ HB101 Competent Cell, and is available through Cosmo Bio. 4) Escherichia co ( Escherichia co )
li ) RR1 Bethesda Research, Inc. is commercially available under the trade name of Library Efficiency ^™ RR1 Competent Cell and is available through Cosmo Bio. 5) pBR322 plasmid Commercially available from Toyobo Co., Ltd. Code No. DNA
-003. 6) pTrc99A plasmid commercially available from Pharmacia. Code No. 27
-5007-01.

7) Salmonella typhimurium ( Sal
monella typhimurium ) IFO14
209 shares Fermentation Research Institute [INSTITUTE FOR F
ERMENTATION, OSAKA (IFO)], No. It is commercially available under the catalog number 14209. This strain was originally a mutant strain prepared from Salmonella typhillium LT-2 strain for use in Ames test (Proc. Natl. Acad. Sci., 7
0: 782-786 (1973)), histidine auxotrophy, membrane mutation rfa , and uvrB that govern excision repair ability.
It has three types of mutations called gene defects. Due to the membrane mutation, the permeability of the chemical substance to the cell membrane is good, and further, the DNA repair ability is impaired by the uvrB deficiency, and therefore the sensitivity to the mutagenic substance is improved. From this fact, this strain was also tested in the mutagenicity test umtest described above using umuD.
It has been used as a host for plasmids containing the C - lacZ fusion gene (Mutation Res., 14).
7: 219-229 (1985)).

[0037] Example 1 1) lexA operator, isolated lexA operator of recA promoter and the recA gene, recA promoter and r
The ecA gene was excised from the chromosomal DNA of the Escherichia coli K-12 strain with Bam HI as a DNA fragment of about 3.1 kb. For cloning, refer to the literature (J. Bact
eriol. , 163: 568-572 (1985)).
And a shotgun method was performed to obtain a plasmid represented by the following formula (2).

Embedded image ( Wherein lexA op, recA pr, Amp and p
BR322 shows the same thing as the above. recA is rec
The A gene is shown. )

That is, the chromosomal DNA of the Escherichia coli K-12 strain was purified, cleaved with Bam HI, and then mixed with pBR322 plasmid for ligation. The ligation solution was mixed with competent cells of Escherichia coli HB101 (cells in a state of incorporating added DNA) prepared by treatment with CaCl ₂ , and transformation was performed. Then, the appropriately diluted bacterial solution was inoculated on an LB plate containing 0.01% methyl methanesulfonate (this compound is a mutagenic substance; hereinafter, sometimes abbreviated as MMS). 37
After culturing overnight at ℃, the colonies appearing on the plate were cultured in a liquid medium, and then the plasmid was purified. The resulting plasmid was digested with Bam HI, was investigated cleavage pattern by Bam HI by agarose gel electrophoresis.
As a result, the pBR322 of Bam HI site about 3.
A 1 kb DNA fragment was incorporated. Furthermore, when the cleavage pattern of the plasmid was examined using two types of restriction enzymes ( Eco RI, Pst I), it was found in the literature (Proc.
tl. Acad. Sci. , 77: 313-317 (1
980), Proc. Natl. Acad. Sci. ，
77: 2611-2615 (1980)), and a DNA fragment having the same size was confirmed.

In order to confirm that the obtained DNA fragment contains the lexA operator, recA promoter and recA gene, the following experiment was conducted. First, the plasmid of the above formula (2) incorporating a Bam HI DNA fragment of about 3.1 kb was originally used as recA.
Recombinant bacteria introduced into Escherichia coli HB101 having no gene [HB101 (pBR322 + recA )]
It was created. As a control, a recombinant bacterium [HB101 (pBR322)] into which only pBR322 was introduced was prepared. These two kinds of recombinant bacteria were cultured in a liquid medium until the absorbance of light having a wavelength of 600 nm (hereinafter referred to as OD ₆₀₀ ) was about 2.4. Then, this culture solution was diluted 10 ⁷ times, and 100 μl of this bacterial solution was inoculated to each LB plate containing an appropriate amount (0 to 2 mM) of methyl methanesulfonate. After culturing at 37 ° C. overnight, colonies appearing on each plate were counted. As a result, HB101 (pBR322) used as a control could not form any colony in the presence of methyl methanesulfonate. On the other hand, HB101 (pBR322 + rec
In A ), the effect of the methyl methanesulfonate concentration on the colony forming ability was not observed in the concentration range of 0 to 2 mM. From the above, the acquired D of about 3.1 kb
It was confirmed that the NA fragment contained the lexA operator, the recA promoter and the recA gene.

[0040] 2) luxAB gene isolation luxAB gene chromosomal DNA of Vibrio Harubei of
From was excised as a DNA fragment of about 4kb by Hin dIII. Cloning is in the literature (Biochemist
ry 23: 3663-3667 (1984)) and performed by the shotgun method to obtain a plasmid represented by the following formula (3).

[Chemical 4] (In the formula, luxAB , Amp and pBR322 are the same as described above.)

That is, chromosome D of Vibrio halveyi
Purification of the NA, was cut by Hin dIII, pBR
Ligation was performed by mixing with 322 plasmid.
The ligation solution was mixed with competent cells of Escherichia coli RR1 prepared by treatment with CaCl _{2 and} subjected to transformation, and then an appropriate amount of the bacterial solution was inoculated on the LB plate. After culturing overnight at 37 ° C., 20 μl of n-decylaldehyde was added to the lid of the plate as a substrate for luminescence, and the colonies were exposed to steam, and luminescent colonies were confirmed. One of these was taken and cultured in liquid to purify the plasmid. When the obtained plasmid was cleaved with a restriction enzyme and the cleavage pattern was examined, it was found that the luxAB gene was correctly integrated in pBR322.

3) Preparation of fusion gene The plasmid of the above formula (2) was cleaved with Bam HI and le
xA operator, recA promoter and rec
A DNA fragment of about 3.1 kb containing the A gene was purified by agarose gel electrophoresis. This DNA fragment is called pT
was introduced into Bam HI site of rc99A plasmid to obtain plasmid represented by the following formula (4).

Embedded image (In the formula, lexA op, recA pr, recA and Amp represent the same as above. PTrc99A represents the nucleotide sequence derived from the plasmid pTrc99A, and P represents the trc promoter.)

The plasmid of the above formula (4) was digested with Pst I to remove the 1.8 kb DNA fragment. The remaining 5.4 kb DNA fragment was purified by agarose gel electrophoresis and subjected to self-ligation to prepare a plasmid represented by the following formula (5). In the plasmid of the following formula (5), Bam HI to Pst I
It was found that the 1.2 kb DNA fragment contained a lexA operator and a recA promoter (Proc. Natl. Acad. Sci., 76:
3144-3148 (1978)).

[Chemical 6] (Wherein, lexA op, recA pr, Amp, pTr
c99A and c are the same as above. )

After cleaving the plasmid of the above formula (5) with Pst I, the cleaved surface was blunted with T4 polymerase and further S
The pTR10 plasmid represented by the following formula (6) was prepared by ligating the al I linker.

[Chemical 7] (Wherein, lexA op, recA pr, Amp, pTr
c99A and c are the same as above. )

In the plasmid of the above formula (3), the luxAB genes required for light emission are Sal I to Pvu II 2.
It is known to be included in the 4 kb portion (J. Ba.
cteriol. , 174: 8094-8101 (19
92)). Therefore, the plasmid of the above formula (3) was added to Pvu
It was cut with II and a Sal I linker was ligated to the cut ends. This was cleaved with Sal I, subjected to agarose gel electrophoresis, and the band portion containing the 2.4 kb DNA fragment was excised to purify the DNA.

Next, the 2.4 kb DNA fragment containing the luxAB gene obtained above and pTR10 of the above formula (6) were used.
The plasmid was cut with Sal I and mixed with dephosphorylated pTR10 and ligated to obtain a plasmid pTRL10 represented by the following formula (7). This plasmid is a novel plasmid with a novel fusion gene containing the lexA operator, the recA promoter and the luxAB gene.

Embedded image ( Wherein lexA op, recA pr, luxAB , p
Trc99A and P have the same meanings as described above. )

[0047] 4) Since pTRL10 recombinant plasmid creating the expression (7) incorporating a fused gene in pTrc99A, even under the condition that no mutagens, tr contained in pTrc99A
Reading from the c promoter into the fusion gene occurs, resulting in the luminescence of the microorganism containing pTRL10. In order to avoid such a reading from the front part of the fusion gene, the fusion gene was excised and introduced into another plasmid. First, the pTRL10 of the formula (7) and Bam HI
It was cut with Hin dIII, consisting of the fusion gene 3.6kb
Was purified. This 3.6 kb DNA fragment was digested with Eco RI and Bam HI to generate pBR32.
It was mixed with 2 and ligated. By this operation, the novel plasmid pMA shown in the above restriction enzyme map (1) is obtained.
S10 was obtained. The literature (Gene 50: 3-40
(1986)), it is known that there is no significant promoter in front of the introduced fusion gene.

5) Preparation of transformant pMAS10 shown in the above restriction enzyme map (1) was transformed into BIO.
Salmonella by electroporation method using -RAD manufactured by Gene Pulser typhim
urium IFO14209 strain and transformed, pMAS10-introduced Salmonella (IFO14
209 (TA1535)) was obtained.

Example 2 Using the Salmonella bacterium introduced with pMAS10 obtained in 5) of Example 1, a mutagenicity test was conducted as follows.
As a mutagenic substance, methyl methanesulfonate (M
MS), mitomycin C (MMC), and 4-nitroquinoline-N-oxide (4NQO). First, 5 ml of Salmonella obtained in Example 1
Was inoculated into LB medium and cultured overnight at 37 ° C. 1 ml of this culture was inoculated into 100 ml of fresh LB medium, and 37
After culturing at 0 ° C. for 2 hours (the OD ₆₀₀ reached around 0.4), 5 ml of the culture solution was dispensed into test tubes. The above-mentioned mutagenic substances were added to these test tubes at various concentrations and incubated at 37 ° C for 2 hours.

10 μl of this culture solution was added to a new LB medium 99.
0 μl was added to make 100-fold dilution, and this diluted culture solution 20
0 μl was used for luminescence measurement. For the luminescence, the diluted culture solution 20 was placed in a cuvette for luminescence measurement in which 10 μl of n-decylaldehyde previously diluted with ethanol 10 times was dispensed.
It was started by adding 0 μl. The amount of light emission was measured for 10 seconds from 5 seconds to 15 seconds after the start of light emission. For the measurement, a luminometer Model 20e (trademark) manufactured by Turner Design Co., Ltd. was used.
5 sec. , Integrate time 10se
c. It was used under the conditions. All the above operations were performed at room temperature. The amount of luminescence was expressed as a measured value of luminescence per OD ₆₀₀ = 1 (measured value of luminometer / OD ₆₀₀ value of stock solution of culture solution). The results are shown in FIGS. 1 to 3 (measurement was performed 3 times, and the average value thereof was taken as the amount of light emission).

As can be seen from FIGS. 1 to 3, MMS, M
MC, 4NQO added concentration is 10μg / l,
In the range of 0.03 μg / ml and 0.03 μg / ml or more, an increase in the amount of luminescence corresponding to the concentration of the mutagen was confirmed. Sensitivity of detection of MMS, MMC, 4NQO by the umu test, which is one of the existing mutagenicity tests (concentration of mutagenic substance exhibiting β-galactosidase activity that is twice the value in the absence of mutagenic substances) ) Are 27, 0.
Since it is 0.05 and 0.08 μg / ml, a sensitivity comparable to this is obtained.

[0052]

[Sequence list]

 SEQ ID NO: 1 Sequence Length: 25 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Genomic DNA Origin Biological Name: Escherichia coli Strain Name: K-12 Sequence Feature symbol: binding-site Location: 1..20 Feature determination method: S Feature symbol: promoter Location: 19..25 Feature determination method: S Sequence TACTGTATGA GCATACAGTA TAATT 25

SEQ ID NO: 2 Sequence length: 2403 Sequence type: Nucleic acid Number of strands: Double-strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Vibrio herveyi Sequence features Features Symbol: cleavage-site Location: 1..6 Feature determination method: S Feature symbol: peptide Location: 137..1201 Feature determination method: S Feature symbol: peptide Location: 1231..2202 Characteristic determination method: S Characteristic symbol: cleavage-site Location: 2398..2403 Characteristic determination method: S Sequence GTCGACTTTA TCGAGCCTGA TTTTGAACAA CTCACCATCG CGACTGTGAA TGAACGTCGC 60 TTGAAAGCGG AAATTGAAAG CCGTACGCGTGAAAGCGG AAATTGAAAG CCGTACAGCAGACT ATG AAA TTT GGA AAC TTC CTT CTC ACT TAT CAG CCA 172 Met Lys Phe Gly Asn Phe Leu Leu Thr Tyr Gln Pro 1 5 10 CCT GAG CTA TCT CAG ACC GAA GTG ATG AAG CGA TTG GTT AAT CTG GGC 220 Pro Glu Leu Ser Gln Thr Glu Val Met Lys Arg Leu Val Asn Leu Gly 15 20 25 AAA GCG TCT GAA GGT TGT GGC TTC GAC ACC GTT TGG TTG CTA GAG CAC 268 Lys Ala Ser Glu Gly Cys Gly Phe Asp Thr Val Trp Leu Leu Glu His 30 35 40 CAC TTC ACT GAA TTT GGG TTG TTA GGG AAT CCT TAT GTT GCT GCC GCA 316 His Phe Thr Glu Phe Gly Leu Leu Gly Asn Pro Tyr Val Ala Ala Ala 45 50 55 60 CAC CTA TTA GGT GCG ACA GAA ACG CTC AAC GTT GGC ACT GCA GCT ATC 364 His Leu Leu Gly Ala Thr Glu Thr Leu Asn Val Gly Thr Ala Ala Ile 65 70 75 GTA TTG CCG ACT GCC CAT CCG GTT CGA CAA GCA GAA GAC GTA AAC CTA 412 Val Leu Pro Thr Ala His Pro Val Arg Gln Ala Glu Asp Val Asn Leu 80 85 90 CTG GAT CAA ATG TCA AAA GGA CGA TTC CGT TTT GGT ATT TGT CGC GGT 460 Leu Asp Gln Met Ser Lys Gly Arg Phe Arg Phe Gly Ile Cys Arg Gly 95 100 105 TTG TAC GAT AAA GAT TTT CGT GTC TTT GGT ACA GAC ATG GAT AAC AGC 508 Leu Tyr Asp Lys Asp Phe Arg Val Phe Gly Thr Asp Met Asp Asn Ser 110 115 120 CGA GCC TTA ATG GAC TGT TGG TAT GAC TTG ATG AAA GAA GGC TTC AAT 556 Arg Ala Leu Met Asp Cys Trp Tyr Asp Leu Met Lys Glu G ly Phe Asn 125 130 135 140 GAA GGC TAT ATC GCG GCG GAT AAC GAA CAT ATT AAG TTC CCG AAA ATC 604 Glu Gly Tyr Ile Ala Ala Asp Asn Glu His Ile Lys Phe Pro Lys Ile 145 150 155 CAA CTG AAT CCA TCG GCT TAC ACA CAA GGT GGT GCT CCT GTT TAT GTC 652 Gln Leu Asn Pro Ser Ala Tyr Thr Gln Gly Gly Ala Pro Val Tyr Val 160 165 170 GTC GCG GAG TCA GCA TCA ACG ACA GAA TGG GCT GCA GAG CGT GGC CTA 700 Val Ala Glu Ser Ala Ser Thr Thr Glu Trp Ala Ala Glu Arg Gly Leu 175 180 185 CCA ATG ATT CTA AGC TGG ATC ATC AAC ACT CAC GAG AAG AAA GCG CAG 748 Pro Met Ile Leu Ser Trp Ile Ile Asn Thr His Glu Lys Lys Ala Gln 190 195 200 CTT GAT CTT TAC AAC GAA GTC GCG ACT GAA CAT GGC TAC GAT GTG ACT 796 Leu Asp Leu Tyr Asn Glu Val Ala Thr Glu His Gly Tyr Asp Val Thr 205 210 215 220 AAG ATT GAC CAC TGT TTG TCT TAC ATC ACC TCC GTC GAT CAT GAC TCA 844 Lys Ile Asp His Cys Leu Ser Tyr Ile Thr Ser Val Asp His Asp Ser 225 230 235 AAT AGA GCC AAA GAT ATT TGC CGC AAC TTC TTG GGC CAT TGG TAC GAC 892 Asn Arg Ala Lys Asp Ile Cys Arg Asn P he Leu Gly His Trp Tyr Asp 240 245 250 TCA TAC GTG AAT GCC ACC AAG ATT TTT GAC GAC TCT GAC CAA ACA AAA 940 Ser Tyr Val Asn Ala Thr Lys Ile Phe Asp Asp Ser Asp Gln Thr Lys 255 260 265 GGT TAC GAC TTC AAT AAA GGT CAA TGG CGT GAT TTT GTG TTG AAA GGC 988 Gly Tyr Asp Phe Asn Lys Gly Gln Trp Arg Asp Phe Val Leu Lys Gly 270 275 280 CAC AAA GAC ACC AAT CGC CGA ATT GAT TAC AGC TAC GAA ATC AAC CCA 1036 His Lys Asp Thr Asn Arg Arg Ile Asp Tyr Ser Tyr Glu Ile Asn Pro 285 290 295 300 GTA GGG ACG CCT GAA GAG TGT ATC GCG ATT ATC CAG CAA GAT ATT GAT 1084 Val Gly Thr Pro Glu Glu Cys Ile Ala Ile Ile Gln Gln Asp Ile Asp 305 310 315 GCG ACG GGT ATT GAC AAT ATT TGT TGT GGT TTT GAA GCA AAC GGT TCT 1132 Ala Thr Gly Ile Asp Asn Ile Cys Cys Gly Phe Glu Ala Asn Gly Ser 320 325 330 GAA GAA GAA ATT ATC GCA TCT ATG AAG CTA TTC CAG TCT GAT GTG ATG 1180 Glu Glu Glu Ile Ile Ala Ser Met Lys Leu Phe Gln Ser Asp Val Met 335 340 345 CCA TAT CTC AAA GAA AAA CAG TAATTAATAT TTTCTAAAAG GAAAGAGAC ATG 1233 Pro Tyr Leu Lys Glu Lys Gln Met 350 355 1 AAA TTT GGA TTA TTC TTC CTC AAT TTT ATG AAT TCA AAG CGT TCT TCT 1281 Lys Phe Gly Leu Phe Phe Leu Asn Phe Met Asn Ser Lys Arg Ser Ser 5 10 15 GAT CAA GTC ATC GAA GAA ATG TTA GAT ACC GCA CAT TAC GTA GAT CAG 1329 Asp Gln Val Ile Glu Glu Met Leu Asp Thr Ala His Tyr Val Asp Gln 20 25 30 TTG AAG TTT GAC ACG TTG GCT GTT TAC GAA AAC CAT TTC TCG AAC AAT 1377 Leu Lys Phe Asp Thr Leu Ala Val Tyr Glu Asn His Phe Ser Asn Asn 35 40 45 GGT GTG GTT GGT GCC CCA CTA ACA GTG GCT GGT TTT TTA CTT GGT ATG 1425 Gly Val Val Gly Ala Pro Leu Thr Val Ala Gly Phe Leu Leu Gly Met 50 55 60 65 ACA AAG AAC GCC AAA GTG GCT TCG TTG AAT CAC GTC ATT ACC ACG CAT 1473 Thr Lys Asn Ala Lys Val Ala Ser Leu Asn His Val Ile Thr Thr His 70 75 80 CAT CCA GTA CGT GTG GCG GAA GAA GCG TGT CTA CTT GAC CAA ATG AGT 1521 His Pro Val Arg Val Ala Glu Glu Ala Cys Leu Leu Asp Gln Met Ser 85 90 95 GAA GGC CGT TTT GCC TTT GGC TTT AGT GAT TGT GAA AAG AGT GCA GAT 1569 Glu Gly Arg Phe Ala Phe Gly Phe Ser Asp Cys Glu Lys Ser Ala Asp 100 105 110 ATG CGC TTC TTT AAT CGA CCA ACG GAT TCT CAG TTT CAG TTG TTC AGT 1617 Met Arg Phe Phe Asn Arg Pro Thr Asp Ser Gln Phe Gln Leu Phe Ser 115 120 125 GAG TGT CAC AAG ATC ATC AAT GAT GCA TTC ACT ACT GGG TAC TGC CAT 1665 Glu Cys His Lys Ile Ile Asn Asp Ala Phe Thr Thr Gly Tyr Cys His 130 135 140 145 CCA AAC AAT GAT TTT TAT AGT TTT CCT AAA ATC TCC GTT AAC CCA CAC 1713 Pro Asn Asn Asp Phe Tyr Ser Phe Pro Lys Ile Ser Val Asn Pro His 150 155 160 GCG TTC ACT GAA GGC GGT CCT GCG CAA TTT GTG AAT GCG ACG AGC AAA 1761 Ala Phe Thr Glu Gly Gly Pro Ala Gln Phe Val Asn Ala Thr Ser Lys 165 170 175 GAA GTG GTT GAA TGG GCG GCT AAG TTA GGG CTT CCA CTC GTG TTT AGA 1809 Glu Val Val Glu Trp Ala Ala Lys Leu Gly Leu Pro Leu Val Phe Arg 180 185 190 TGG GAC GAC TCA AAC GCT CAA AGA AAA GAA TAC GCC GGT TTG TAC CAC 1857 Trp Asp Asp Ser Asn Ala Gln Arg Lys Glu Tyr Ala Gly Leu Tyr His 195 200 205 GAA GTT GCT CAG GCA CAT GGT GTC GAT GTT AGT CAG GTT CGA CAC AAG 1905 Glu Val Ala Gln Ala His Gly Val Asp Val Ser Gln Val Arg His Lys 210 215 220 225 CTG ACG CTG CTG GTC AAC CAA AAT GTA GAT GGT GAA GCA GCA AGG GCA 1953 Leu Thr Leu Leu Val Asn Gln Asn Val Asp Gly Glu Ala Ala Arg Ala 230 235 240 GAA GCT CGC GTG TAT TTG GAA GAG TTT GTC CGT GAA TCT TAC TCA AAT 2001 Glu Ala Arg Val Tyr Leu Glu Glu Phe Val Arg Glu Ser Tyr Ser Asn 245 250 255 ACC GAC TTT GAG CAA AAA ATG GGA GAG CTG TTG TCA GAA AAT GCC ATC 2049 Thr Asp Phe Glu Gln Lys Met Gly Glu Leu Leu Ser Glu Asn Ala Ile 260 265 270 GGT ACT TAT GAA GAA AGT ACT CAG GCA GCG CGA GTT GCG ATT GAG TGT 2097 Gly Thr Tyr Glu Glu Ser Thr Gln Ala Ala Arg Val Ala Ile Glu Cys 275 280 285 TGT GGT GCC GCG GAC CTA TTG ATG TCT TTT GAG TCG ATG GAA GAT AAA 2145 Cys Gly Ala Ala Asp Leu Leu Met Ser Phe Glu Ser Met Glu Asp Lys 290 295 300 305 GCG CAG CAA AGA GCG GTT ATC GAT GTG GTA AAC GCC AAC ATC GTC AAA 2193 Ala Gln Gln Arg Ala Val Ile Asp Val Val Asn Ala Asn Ile Val Lys 310 315 320 TAC CAC TCG TAACGTTTAA CTGATGCTGA AGGGGCAGCG ATGCCCCTTA 2242 Tyr His Ser TATCACCAT T CTTTTCGCCG ATAGCGCTAA CTAATAGAGG CATTTATATG GACGTACTTT 2302 CAGCGGTTAA GCAGGAAAAC ATCGCAGCGA GCACAGAAAT CGATGACTTG ATTTTCATGG 2362 GAACTCCTCA GCAATGGTCA TTGCAGGAAC AAAAACAGCT G 2403

[Brief description of drawings]

FIG. 1 is a graph showing the results of Example 2.

FIG. 2 is a graph showing the results of Example 2.

FIG. 3 is a graph showing the results of Example 2.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location (C12N 1/21 C12R 1:19) (C12N 1/21 C12R 1:42) (C12N 1/21 C12R 1:63)

Claims (5)

[Claims] 1. A lux- encoding lux downstream of an operator to which a lexA protein as a repressor binds and a promoter of a recA gene.
A fusion gene characterized by binding to the AB gene. 2. A lux- encoding lux downstream of an operator to which a lexA protein as a repressor binds and a promoter of a recA gene.
A recombinant plasmid having a fusion gene to which the AB gene is linked. 3. A luciferase-encoding lux downstream of an operator to which a lexA protein as a repressor binds and a promoter of a recA gene.
A recombinant plasmid pMAS10 having a fusion gene to which the AB gene is bound, which is represented by the following restriction enzyme map (1). Embedded image (In the map, lexA op is the operator to which the lexA protein binds, recA pr is the promoter of the recA gene, luxAB is the luxAB gene, Amp is the ampicillin resistance gene, and pBR322 is the plasmid pB.
The base sequence derived from R322 is shown. ) 4. A transformant introduced with the recombinant plasmid according to claim 2 or 3. 5. A mutagenicity test method, which comprises culturing the transformant according to claim 4 in a medium containing a sample, and then adding a luminescent element which is a substrate of luciferase to cause luminescence.